Biological fluids usually contain a mixture of several proteins, and one of the major achievements of modern biochemistry is to have devised methods for their separation. Best example if blood plasma or serum, where methods are available for identification and separation of at least 25 major protein components (Schultze and Heremans: Molecular Biology of Human Proteins, Elsevier, 1966). Other examples of naturally occurring complex proteins mixtures is milk or whey, urine, spinal fluid, egg white, etc.
For the purpose of the present disclosure, it is helpful to define the following protein nomenclature, the classification being based on their solubility in a variety of solvents: (1) albumin is the major protein component of plasma, serum, and egg white, and is characterized by being soluble both, in half-saturated ammonium sulfate and in distilled water; (2) globulins are those proteins of plasma or other biological fluids which precipitate in half-saturated ammonium sulfate; (3) euglobulins are those globulins which are not only precipitated in half-saturated ammonium sulfate, but also in deionized water, as they apparently need some salts to be soluble. Obviously, this classification is arbitrary, though widely used in protein chemistry, as the solubility of all proteins depends also on the pH of the solution, temperature, and other solutes present, such as alcohol; (4) euglobulin-like materials; the term "euglobulin-like" is used herein for those proteins which precipitate in deionized aqueous solutions only in presence of various amounts of alcohol. These proteins are not true euglobulins, being soluble in deionized water in absence of alcohol, yet they possess some of the characters of the euglobulins, being solubilized by even low concentrations of salts.
It will further help to define, for the purposes of this invention, the following electrical membrane processes:
1. Electrodialysis is primarily used for the desalting of aqueous solutions. Uusally, this is accomplished by means of ion-selective membranes, said membranes allowing preferential passage of either positively or negatively charged ions, as described in a variety of patents including U.S. Pat. Nos. 2,694,680, 2,848,402, 2,860,091, 2,777,811. The usefulness of this technique for the separation of proteins has not previously been recognized. Ion-selective membranes can also be substituted by essentially electrically neutral membranes, with inclusion of polyelectrolytes into certain compartments, these polyelectrolytes becoming polarized along the membranes under the influence of an electrical field, thereby conveying to the neutral membranes an element of ion-selectivity as taught in U.S. Pat. Nos. 3,677,923 and 3,725,235. In other electrical membrane processes, discussed in the following two sections, some electrodialysis is unavoidably superimposed to other effects sought, being a direct result of the passage of electrical current. For the purpose of the present invention, the term electrodialysis will be reserved to these electrical processes, the primary purpose of which is desalting, preferentially accomplished either with ion-selective membranes or with the use of polyelectrolytes. PA1 2. Electrodecantation is an electrical process for concentration and separation of a variety of colloids including proteins as taught in U.S. Pat. Nos. 2,057,156, 2,292,608, 2,762,770, 2,800,448, 2,801,962. These teach devices which contain a multitude of essentially electrically-neutral membranes in a parallel array, the colloids or proteins accumulating under the influence of the electrical current or fields in the immediate neighborhood of said membranes and are decantable along said membranes as a result of density gradients. An analogous method is sometimes referred to as electrophoresis-convenction (in U.S. Pat. No. 2,758,966), where usually only a single pair of electrically neutral membranes is employed for the purpose of creating electrodecantation in the protein solution. These techniques have been widely used for protein fractionation, principally for preparation of gamma globulins, these proteins of plasma being isoelectric and not decanting. This technique has not been taught for the preparation of serum albumin, the most mobile of plasma proteins (in terms of electrophoresis). Among the objectives of the present disclosure is to teach utilization of such techniques for fractionation for the preparation of serum albumin. PA1 3. Forced-flow electrophoresis includes devices similar to those for electro-decantation, which also utilize a parallel array of electrically neutral membranes, but the partitions are located between adjacent pairs of membranes. Such partitions permit better control of flow patterns within the apparatus, and also act as diffusion barriers. Two such electrophoresis devices are described in U.S. Pat. Nos. 2,878,178 and 3,079,318, and the technique also is described as "forced-flow electrophoresis" by M. Bier; "Electrophoresis", Academic Press, 1959, page 295. PA1 1. the process of fractionation of proteins including plasma or plasma fractions, comprising causing the precipitation of an euglobulin fraction by means of electrodialytic desalting under controlled conditions of temperature, pH, and conductivity and recovering the dialysate. PA1 2. the process of fractionation of proteins including plasma or plasma fractions, comprising precipitation of an euglobulin-like fraction by means of electrodialytic desalting under controlled conditions of temperature, pH, conductivity, and alcohol content. PA1 3. process of fractionation of proteins including plasma or plasma fractions, comprising preparation of an euglobulin-like precipitate by electrodialytic desalting under controlled conditions of temperature, pH, conductivity, and alcohol content, followed by selective dissolution of albumin-enriched fraction by re-adjustment of temperature, pH, conductivity, or alcohol content. PA1 4. process of plasma fractionation, comprising the steps including a first precipitation by alcohol, a second step of electrodialytic desalting of the supernatant of said first precipitation, said second step causing precipitation of an euglobulin-like fraction, an elective third step comprising selective dissolution of an albumin rich fraction from said euglobulin-like precipitate, and a last step of alcohol precipitation of an albumin-rich fraction from the combined supernatants of electrodialytic desalting step or, alternatively from the combined supernatants of the second step and the elective third step of fractionation. PA1 5. process of improving fractionation of protein mixtures by electrodecantation or forced-flow electrophoresis, comprising the reduction of their salt content through prior electrolytic desalting, said desalting causing also precipitation of euglobulins or euglobulin-like materials. PA1 6. process of improving fractionation of protein mixtures by electrodecantation or forced-flow electrophoresis, comprising the reduction of their salt content through prior desalting, and subsequent addition of a buffering salt, said buffering salt being an ampholyte such as glycine, said desalting causing also precipitation of euglobulins. PA1 7. process of plasma fractionation, comprising a first precipitation by alcohol, a second step of electrodialytic desalting of the supernatant of said first precipitation, said second step causing precipitation of an euglobulin-like fraction from said euglobulin-like precipitate, and a last step comprising the selective concentration of an albumin-rich fraction by means of electrodecantation of forced-flow from the supernatant of the desalting step or alternatively the combined supernatants from the desalting and the elective third step of fractionation. PA1 8. products of manufacture suitable for use as plasma expander, and comprising at least 90% of albumin, obtained by above processes, in particular by processes 4 or 7.
The processes of electrodecantation and forced-flow electrophoresis are similar in principle and results and for purposes of convenience will be often referred to herein as electrofield separations. As set forth below they may be used interchangeably.
The most important protein products of commerce are those obtained from human or animal plasma or serum. Two such proteins, serum albumin and gamma globulins, either from human or animal origin will be used as the principal examples but the scope of this invention can also be applied to other biological fluids or other proteins without modification. The present commercial methods of obtaining these fractions are based on alcohol fractionation, a process developed by Cohn et al and described in U.S. Pat. Nos. 2,390,074, 2,770,6l6. This technique is essentially based on sequential precipitation of various protein fractions by alcohol, under controlled conditions of temperature, alcohol content, pH, and salt content as summarized by C. A. Janeway, Adv. in Internal Med. 3, 295, 1949. This technique requires a large installation, the yield of certain fractions is low, it requires prolonged exposure of proteins to high alcohol content, which has a denaturing effect on some protein fractions. The technique is also limited to production of certain fractions of plasma only; other protein fractions are not recoverable in sufficient states of purity.